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					INTRODUCTION

The West Nile Virus is a mosquito-borne disease, and infects an astonishingly wide
variety of wildlife and domestic animals, in addition to occasionally causing disease in
humans. Scientific investigators are constantly looking for new mosquitoes, as well as
reviewing previously identified specimens for new information or identifying
characteristics. Mosquitoes can be an annoying, serious problem in man’s domain. They
interfere with work and spoil hours of leisure time because of its ability to transmit
disease. There are over 2500 different species of mosquitoes throughout the world.
Around 200 species occur in the United States with 55 species documented in Indiana, all
of which live in specific habitats, exhibit unique behaviors, and bite different types of
animals. There are approximately 12 to 15 species that are of concern to the health of
humans. The mosquito Genus Culex, is the species responsible for carrying the West
Nile Virus. To control the spread of the West Nile Virus, Floyd County has adopted a
mosquito surveillance program to monitor and also help to reduce mosquito harboring.
WEST NILE VIRUS
The West Nile Virus has become increasingly prevalent to the United States. The first
human isolate of the virus was obtained from a woman in the West Nile District of
Uganda in 1937. The virus became recognized as a cause of severe human meningitis or
encephalitis, inflammation of the spinal cord and brain, in elderly patients during an
outbreak in Israel in 1957. Equine encephalitis was first reported in the early 1960’s in
Egypt and France. The virus finally made its way to North America in 1999, reported as
encephalitis in humans and horses. Since its introduction into the United States in 1999,
the West Nile virus has spread to almost every state showing up in horses, birds,
veterinary and mosquitoes. Alaska and Hawaii have yet to report a positive case of the
West Nile Virus.

Most people infected with the West Nile
Virus often experience no signs or symptoms.
About 20% of people develop a mild infection
called the West Nile fever characterized by a
skin rash, headache, fever, diarrhea, nausea,
vomiting, and swollen lymph glands. In less       Figure 1. West Nile Virus Transmission

than 1% of infected people, the virus causes a more serious neurological infection usually
lasting for three to six days or possibly longer. The neuron-invasive form occurs most
often in people over age 50 and some immuno-compromised persons, but can occur at
any age in healthy individuals.

ECOLOGY OF WEST NILE VIRUS
The emergence of the West Nile Virus in human populations is associated with prolonged
periods of hot, dry weather that is often followed by a significant rain event. Under these
conditions, infection seems to shift from birds to humans. In the United States, wild
birds, especially crows and jays, are the main reservoir of the West Nile Virus, but the
virus is actually spread by certain species of mosquitoes. When a mosquito bites a bird
infected with the West Nile Virus, the virus enters the mosquito’s bloodstream and
circulates for a few days before setting into the salivary glands. When the infected
mosquito bites an animal or human, the virus then enters the host’s bloodstream, where it


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can cause serious illness. Although the exact mechanism of illness is unknown, the West
Nile Virus probably enters the host’s bloodstream, multiplies and moves on to the brain,
crossing the blood-brain barrier – a barrier that separates the blood from the central
nervous system. Once the virus crosses that barrier and infects the brain or its linings, the
brain tissue becomes inflamed and symptoms arise. The West Nile Virus transmission
occurs mostly during warm weather, when mosquito populations are active. The
incubation period – the period between when you’re bitten by an infected mosquito and
the appearance of signs and symptoms of the illness – ranges from two to 14 days. The
cyclic process of bird to mosquito means the virus can travel a great distance. When the
West Nile Virus travels to a larger mammal, such as a horse or a human, there is less
chance that the virus can be delivered back to the mosquito due to a low presence of the
virus in their bloodstream. In rare cases, it is possible for the West Nile Virus to spread
through other routes, including organ transplantation, blood transfusions, mother to
unborn child, as well as breast-feeding.

MOSQUITO BIOLOGY
Most mosquitoes lay their eggs on the surface of fresh or stagnant water. The water may
be in tin cans, barrels, horse troughs, ornamental ponds, swimming pools, puddles,
creeks, ditches, catch basins, or marshy areas. The Culex mosquitoes usually lay their
eggs at night over a period of time sticking them together to form a raft of anywhere from
100 to 300 eggs. In addition, a female mosquito may lay a raft of eggs every third night
during its life span.

Tiny mosquito larvae will emerge from the eggs within 24-48
hours almost in unison. The mosquito larvae live in the water
from 4 to 14 days depending on the water temperature. The
larvae must come to the surface frequently to obtain oxygen
through a breathing tube called a siphon. Their mouths filter
anything small enough to be eaten to nourish them, including         Figure 2. Life Cycle of Mosquito

algae, plankton, fungi, and bacteria. The larvae are able to break the surface of water to
retrieve oxygen and continue to thrive in polluted or shallow water while maturing
without the threat of predators. During growth, the larvae shed its skin four times,
turning into the pupae stage.

Mosquito pupae live in the water from 1 to 4 days, depending upon species and
temperature. The pupae are lighter than water and therefore are able to float at the
surface. They also take oxygen through two breathing tubes, but do not ever eat. The
metamorphosis of the mosquito into an adult is completed within the pupae case. The
adult mosquito splits the pupae case and will then emerge to the surface of the water
where it rests until its body dries and hardens.

Only female mosquitoes require a blood meal and will bite animals and birds to do so.
Stimuli that influence biting (blood feeding) include a combination of carbon dioxide,
temperature, moisture, smell, color, and movement. Acquiring a blood meal is essential
for egg production, but both male and female mosquitoes are nectar feeders. Of the
female mosquitoes capable of blood feeding, human blood meals are seldom first or
second choices, while horses, cattle, smaller mammals and/or birds are also preferred.


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The Culex species are painful and persistent biters, but also prefer to attack at dusk and
after dark. These mosquitoes usually live only a few weeks during the warm summer
months. The females that emerge in late summer search for sheltered areas where they
can hibernate until spring. Warm weather brings them out again in search of water on
which they lay their eggs. To help reduce mosquito bites during this time, wear long
sleeve shirts, tuck pant legs into socks, as well as, apply DEET, Picaridin, or other
repellents. When applying repellent on small children, pay special attention to the
concentrations listed on the labels.




Figure 3. Home Prevention


PREVENTION AND MONITORING
An efficient way to control mosquitoes is to find and eliminate their breeding sites.
Eliminating large breeding areas such as swamps or sluggishly moving streams or ditches
may require a community-wide effort. This is usually a task for your organized mosquito
control program. The best prevention of mosquito breeding is to remove their habitat by
eliminating tin cans, old tires, buckets, unused swimming pools, or other containers that
collect and hold water around the home. By emptying or changing the water in bird
baths/fountains, sweeping away water that can collect in low areas at least once a week,
stocking ornamental ponds with fish that eat mosquito larvae and pupae, as well as
eliminating tall grass or overgrown foliage will help to destroy potential mosquito
habitats.
     “Mosquito Dunks”
Bacillus thuringienis subspecies israelensis strain EG2215, or B.t.i., which are also
known as mosquito dunks or briquettes, are designed for mosquito control. The
briquettes are applied to areas such as drainage basins, irrigation ditches, roadside
ditches, flood water, standing ponds, woodland ponds, snow melt pools, pastures, and
storm water retention areas. B.t.i. is a bacteria toxin that infects the mosquito larvae.
When the briquette is dropped into water, it will dissolve and then eventually fall to the
bottom. The B.t.i. produces a protein crystal that is toxic to mosquito larvae. The protein
crystal disrupts the lining of the mosquito larvae’s intestine, causing it to eventually die.
The briquette will last roughly thirty days and cover an area of 100 square feet. The B.t.i.
briquettes are only toxic to mosquitoes and midge insects. There are no documented
health effects to the environment when used in suggested areas.




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   Mosquito Traps
Once a female mosquito has received a blood meal to nourish her
eggs, the next step is to search for an ovipositor site. The CDC
Gravid Trap is an attractive location for the mosquito’s eggs (fig.
4). This trap is baited with an infusion of yeast, straw/hay, and
water. Then the solution is poured into a small tub and a
mosquito net is draped over a cylindrical tube placed over that
solution. Inside the tube a motorized fan, powered by a 0.6V
battery, has the ability to funnel mosquitoes into the net that are
near the base. The CDC CO2 baited light trap is also effective in
trapping mosquitoes. The object of the trap is to attract
mosquitoes that are searching for a blood meal.
                                                                            Figure 4
METHODS
LOCATION
The sites tested for the 2007 monitoring season were developed through call-in
complaints from the public to the Floyd County Health Department. As calls came in,
documentation of the sites were made. First, each site is assessed to explore possible or
potential sources of mosquito breeding or harboring. Taking into account the possibility
of theft, wind, or flood, the site is then observed for possible trap locations. The location
for a trap should not be in an area with high amounts of sun exposure because of the
possibility of the solution evaporating. Next, search for an environment where the female
would desire ovipositor. Urban areas are ideal locations to trap for Culex spps., a
container-breeding species.

TRAPS
Once an ideal trap location is discovered, the CDC Gravid
Trap is prepared for use. The yeast/hay infusion, also
known as bracken water, is to be used in the trap but must
be created in advance for fermentation to take place. This
water solution has a strong smell to attract the female
mosquito to the trap. Each infusion is fermented for a
minimum of two days. Typically the infusion is prepared
near the end of the week for fermentation to take place over
a period of 3 to 4 days. A five-gallon bucket is used to
compile the straw/hay infusion with a few pinches of                         Figure 5
Active Dry Yeast (fig. 5). Lastly, the bucket is filled with water.

At the trapping site, the container is filled with the bracken water to a level that will leave
room between the base of the column and the water. The column is placed over the
container and checked for stability. The net is then placed over the column so it is
straight and is not easily off set. The battery is then attached firmly to the motor. The
fan is observed to be sure there are no obstructions and is moving freely.

COLLECTION AND SEPARATION OF SPECIES
One trap is set in each location. The traps stay at each site for three to four nights,
replacing the mosquito filled net with an empty net, as well as, checking the bracken


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        water level. If needed, more bracken water is added to the trap while making sure to
        leave enough room between the base of the column and the water level. The battery
                                             needs to be charged every couple days to refrain from becoming
                                             weak and allowing mosquitoes to escape past a slow moving fan.
                                             Once the nets are collected from the trapping sites, they are then
                                             placed into a freezer. The nets only need 3 to 4 hours to freeze in
                                             order to kill the mosquitoes, but can be kept over night if desired.
                                             After the freeze, the nets are then opened and the mosquitoes are
                                             examined under a microscope to separate them by species. The
                                             numbers of each species from each location are then recorded
                                             into a database. Mosquitoes are placed into baggies and labeled
                                             with the location and the types of mosquitoes found. The baggies
                                             are then sent to Mike Sinsko, PhD., head of the Entomology
                                             Laboratory in Indianapolis, for further testing of the West Nile
Figure 6. Emptying the nets. Courier Journal Virus in those specific mosquitoes.
Louisville



        PROCEDURE FOR POSITIVE WNV
        The Floyd County Health Department is notified via email, once the laboratory in
        discovers mosquitoes that have been identified as positive. The notification indicates the
        specific site from which the positive mosquito was collected. The site is then observed
        and assessed as a source of the mosquito and sprayed by a licensed pesticide applicator.

        As humans contract the disease, hospitals will notify the health department. The health
        department will then do assessments to identify the potential threat of mosquitoes with
        each possibility being carefully considered. If locations are in the Floyd County area,
        assessments are done for each site. A Gravid Trap is then set near the possible mosquito
        sources. If the mosquitoes are identified positive for the West Nile Virus, steps will then
        be followed to have the area sprayed by a licensed pesticide applicator.

        RESULTS
                                                       DATA
                         Days Tested                                               46
                       Total Traps Set                                             72
                    New Albany City Limits                                         68
                      Outside City Limits                                          4

                                 TOTAL COUNT OF EACH SPECIES TRAPPED 2007
                     Culex Pipien/resutan                                        3299
                      Aedes Albopictus                                            242
                    Ochlerotatus Triseritus                                         9
                    Ochlerotatus Japonicus                                          3
                          Anopheles                                                 6
                      Aedes Grossbecki                                              5
                       Aedes Unknown                                               38
                          Unknown                                                   5



                                                          5
                                      POSITIVE West Nile Virus
       Mosquito Pools (sprayed locations)                         2
                Human Cases                                       1

                                 SPECIES & ABUNDANCE (%)
             Culex Pipien/resutan                                 91%
              Aedes Albopictus                                     7%
            Ochlerotatus Triseritus                               0.3%
            Ochlerotatus Japonicus                               0.08%
                  Anopheles                                       0.2%
              Aedes Grossbecki                                    0.1%
               Aedes Unknown                                       1%
                  Unknown                                         0.1%


NATIONAL DATA
The following map of the United States gives information about the West Nile Virus in
the United States, as of November 6, 2007. The Center for Disease Control (CDC) has
been recording the positive cases of the virus in the U.S. since 1999.

         2007 West Nile Virus Activity in the United States




                     2006                                        2005




                     2004                                        2003




                                                 6
                  2002                                          2001




                  2000                                          1999




STATE DATA
             Indiana Counties with Positive West Nile Virus Mosquito Pools
                   Adams – 2                               Grant – 2
                    Allen – 1                            Hamilton – 12
                    Clark – 4                             Hancock – 1
                  Delaware – 7                            La Porte – 2
                   Elkhart – 1                             Lake – 11
                    Floyd – 2                             Marion – 63
                  Marshall – 1                           St. Joseph – 2
                   Noble – 1                           Vanderburgh – 6
                   Porter – 1                               Vigo – 2
                                                          Whitley – 2



                   Mosquito Pool Positive Counties in Indiana (Fig.9)




                                           7
                       Human Positive Counties in Indiana (Fig. 10)




DISCUSSION

THREAT OF WNV
As of November 6, 2007 there have been 92 recorded deaths from the West Nile Virus in
the United States. In recent years, there have been as many as 284 deaths recorded in one
year. Since 1999, the virus has spread across the United States affecting the health of
humans and their environment in lethal ways. There has been a reduction in the number
of mosquito pools, as well as, humans being identified as positive for the West Nile Virus
since last year. This could be the result of many variables. Weather can play a major
part of transmitting the virus due to birds carrying the virus to wetter climates and back
again when the seasons change. It is a well known fact that the West Nile Virus is a
threat to the state of Indiana, as well as, the United States. Reducing habitats around
homes and taking precautions when outdoors will help reduce mosquito breeding
grounds.

RESOLUTIONS
The Floyd County Health Department is affecting the lives of residents in a positive way
through mosquito control. One resident had over 360 mosquitoes in a gravid trap from
one night, although the mosquitoes tested at this residence were not positive for the West
Nile Virus. The problem was resolved with the help of the city of New Albany’s
Engineering Offices having the area sprayed by a licensed pesticide applicator. Media
outlets, distributing brochures, and word of mouth have been the most helpful ways of
getting the message across to the public about how to reduce the number of mosquito
breeding grounds throughout the community. The local government is also joining in the
effort to reduce mosquito populations and is currently in the process of correcting the
city’s sewage and drainage systems that have been the locations of several breeding sites.

ERRORS
While other species are known to carry the West Nile Virus, the Culex pipien/restuan
species has been the mosquito largely responsible for the spread of the virus. The Culex
species, more so than any other species, has been calculated to have an overwhelming
presence in Floyd County, IN. Information was not given as to what type of species


                                            8
carried the virus when positive results were found. Mosquitoes of Indiana was a
reference used to identify the mosquitoes that were later sent to the entomology lab in
Indianapolis. The Internet was also a resource used to confirm some of the mosquitoes,
as some were difficult to identify due to wear and tear on the mosquito. The mosquitoes
that could not be placed into a Genus or given a species were reported as unknown in the
data section. Due to the difficulty of distinguishing between the pipien and restuan
species, they were grouped together in the report under the Genus Culex. As stated in the
methods section, sites are assessed and traps are set due to complaints from the residents
of Floyd County.

OUTLOOK
The 2007 sites which resulted in positive and large return of mosquito pools will need to
be monitored throughout the 2008 mosquito season to remain proactive in the prevention
of mosquito harboring. To better understand which species are considered local to the
Floyd County Area, a permanent trap in those areas would be able to give us more useful
data. Continued monitoring, trapping, and spraying is important to control the mosquito
population, as well as, dealing with the threat of the West Nile Virus that can be deadly to
our elderly and infant children.




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10
References:

Bodle, Ethan. “West Nile Virus.” Medical Ecology Online. 2004. Mailman School of
Public Health, Columbia University. December 2007.
<http://www.medicalecology.org/diseases/westnile/westnile.htm>.

Bravo, Alejandra, et al. “Mode of action of Bacillus thuringiensis Cry and Cyt toxins and
their potential for insect control.” Toxicon 49.4 (2007): 423-35.

“CDC: West Nile Virus – Background: Virus History & Distribution.” Centers for
Disease Control & Prevention Online. 2004. Division of Vector-Borne Infectious
Diseases. December 2007.
<http://www.cdc.gov/ncidod/dvbid/westnile/background.htm>.

“Did You Know?...Healthy Wetlands Devour Mosquitoes.” Indiana Department of
Natural Resources Online. Division of Fish & Wildlife. December 2007.
<http://www.in.gov/dnr/fishwild/publications/inwetcon/hlywet.pdf>.

“Equine West Nile.” Equine West Nile Online. 2007. Fort Dodge Animal Health.
December 2007. <http://www.equinewestnile.com>.

Hill, Catherine A., Caitlin Shaunnessey, and John McDonald. “The Biology and Medical
Importance of Mosquitoes in Indiana.” Purdue University Online. 2006. Purdue
University, Department of Entomology Cooperative Extension Service. December 2007.
<http://www.entm.purdue.edu/Entomology/ext/targets/e-series/EseriesPDF/E-242/pdf>.

“Mosquito Prevention.” Fight The Bite Colorado Online. 2007. Tri-County Health
Department. December 2007.
<http://www.fightthebitecolorado.com/mosquitoprevention/htm>.

Siverly, R.E. Mosquitoes of Indiana. Indiana: Indiana State Board of Health, 1972.

“West Nile Virus: Risk Factors.” Mayo Clinic Online. 2007. Mayo Foundation for
Medical Education & Research. December 2007. <www.mayoclinic.com/health/west-
nile-virus/DS00438/DSECTION=4>.

Acknowledgments:
I would like to express my appreciation to the Floyd County Health Department for
providing me with the equipment, as well as, the opportunity to be a part of the mosquito
surveillance program during the 2007 season. I would also like to thank Dominique
McCowen for training me and introducing me to the mosquito program.

In particular, I would like to thank Dr. Mike Sinsko, head of the state Entomology Lab in
Indianapolis, for answering my questions about West Nile and for also testing the
mosquitoes I sent for the virus. In addition, I would also like to thank Dr. David Taylor
for allowing me to use his microscope to identify the different mosquito species.



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